ANALYSIS OF THE FUNCTION OF THE NODAL GENE DURING EMBRYONIC DEVELOPMENT

我们的分析导致胚胎停滞的小鼠突变原肠胚形成之前，导致识别和分离的nodal，一个基因编码的TGF-β超家族的分泌信号分子的成员。我们发现，在体外合成的nodal mRNA注入青蛙或鱼胚胎，或青蛙胚胎与重组nodal蛋白的治疗，重定向细胞中胚层的命运，这表明nodal作为中胚层诱导因子在正常小鼠发育。我们还表明，nodal在胚胎发育后期，在左右（L/R）体轴的建立过程中具有重要作用。所有的脊椎动物都有特征性的和保守的L/R内脏不对称，例如左侧心脏。最近，来自几个不同实验室的研究已经开始解释胚胎L/R轴是如何建立的。我们发现，nodal基因是在正常形态学L/R不对称出现之前，鸡胚中沿L/R轴沿着不对称表达的少数基因之一。这种不对称表达在进化上是保守的：nodal在相似的阶段和位置在小鼠胚胎中不对称表达，就像非洲爪蟾nodal相关基因-1（Xnr-1）在青蛙胚胎中一样。在鸡的进一步实验中，对胚胎进行操作，使nodal在身体的左右两侧对称表达，或者根本不表达nodal。在这两组实验中，大约一半的胚胎发育出了右侧心脏。这些结果表明，nodal不是心脏形成所必需的，相反，它的不对称表达是心脏发育具有特征极性所必需的。我们研究了内脏逆位（iv）小鼠突变体中的淋巴结表达，其中L/R发育是随机的（大约一半的iv小鼠具有正常的心脏位置，而另一半具有反转的心脏位置）。在单个静脉注射胚胎中，发现结节表达是对称的（两侧或两侧都没有），或不对称的（仅在左侧或仅在右侧）。因此，iv基因的突变通过使nodal表达的位置随机化来干扰L/R发育。最近，我们一直在研究另外4种显示L/R发育改变的突变体。这些包括Ft、Mgat、nt和SIL基因座处的突变。我们分析了所有四个菌株中的nodal表达，并再次发现改变的模式：Mgat和nt是随机的，如iv，而所有SIL和Ft胚胎在两侧都表达nodal。目前，我们正在更详细地分析iv，SIL和Mgat突变体，以了解为什么每种突变体对nodal表达的影响如此不同。与iv相反，我们发现Mgat和SIL突变体中胚胎结构沿着中部存在缺陷，但缺陷的时间和性质不同。SIL突变体中的一个独特发现是缺乏声刺猬（shh）信号转导途径的活性，这是许多发育过程的重要组成部分。我们现在正在研究SIL在shh通路中的作用，以及是否双侧对称的结表达是由于shh信号传导中断、中线结构缺陷或两者兼而有之。这些结果进一步支持的关键作用，节点在解释和中继L/R图案信息的脊椎动物。- 动物模型，细胞增殖，发育，胚胎干细胞，遗传学，生长因子，原位杂交，蛋白质折叠，受体，-既不是人类受试者也不是人类组织

Our analysis of a mouse mutation causing embryonic arrest prior to gastrulation led to the identification and isolation of nodal, a gene encoding a member of the TGF-beta superfamily of secreted signaling molecules. We found that injection of in vitro synthesized nodal mRNA into frog or fish embryos, or treatment of frog embryos with recombinant nodal protein, redirected cells to a mesodermal fate, suggesting that nodal acts as a mesoderm inducing factor during normal mouse development. We have also shown that nodal has an important role later in embryonic development, during the establishment of the left- right (L/R) body axis. All vertebrates have characteristic and conserved L/R visceral asymmetries, for example the left-sided heart. Recently, studies from several different labs have begun to explain how the embryonic L/R axis is established. We showed that nodal is one of a small number of genes expressed asymmetrically along the L/R axis in the chick embryo before the appearance of normal morphological L/R asymmetry. This asymmetric expression has been evolutionarily conserved: nodal is asymmetrically expressed in mouse embryos at similar stages and locations, as is Xenopus nodal related-1 (Xnr-1) in frog embryos. In further experiments in the chick, embryos were manipulated so that nodal was expressed symmetrically on both the left and right sides of the body, or so that nodal was not expressed at all. In both sets of experiments, approximately half the embryos developed a right-sided heart. These results have shown that nodal is not required for the heart to form, rather its asymmetric expression is necessary for the heart to develop with characteristic polarity. We examined nodal expression in the situs inversus viscerum (iv) mouse mutant in which L/R development is randomized (approximately half of iv mice have normal heart placement while half have reversed heart location). In individual iv embryos, nodal expression was found to be either symmetric (on both sides or on neither side), or asymmetric (on the left side only or on the right side only). Thus, mutations in the iv gene disturb L/R development by randomizing the location of nodal expression.More recently, we have been studying 4 additional mutants that show altered L/R development. These include mutations at the Ft, Mgat, nt and SIL loci. We analyzed nodal expression in all four strains and again found altered patterns: Mgat and nt were random like iv whereas all SIL and Ft embryos expressed nodal on both sides. Currently, we are analyzing iv, SIL and Mgat mutants in greater detail to understand why each affects nodal expression so differently. In contrast to iv, we found defects in embryonic structures along the middle of the embryo in Mgat and SIL mutants, but the timing and nature of the defects were different. A unique finding in SIL mutants was the lack of activity of the sonic hedgehog (shh) signal transduction pathway, an essential component of a number of developmental processes. We are now studying the role of SIL in the shh pathway and whether bilateral symmetry of nodal expression results from disrupted shh signaling, defective midline structures or both. These results further support the critical role nodal plays in interpreting and relaying L/R patterning information in vertebrates. - Animal models, cell proliferation, Development, embryonic stem cells, Genetics, growth factors, in situ hybridization, protein folding, receptors, - Neither Human Subjects nor Human Tissues